The process by which cellular replication occurs is complex, involving many steps and numerous factors including regulatory, cytoskeletal and polymerization proteins. However, some early steps in this complex process are known to be essential, for example, initiation of DNA replication.
In higher eukaryotes, DNA replication is thought to be initiated at many origins of replication present on chromosomal DNA. By virtue of homology to known viral origins of replication, some highly repeated eukaryotic origins of replication have been identified and sequenced (Jelinek et al. 1980 Proc. Natl. Acad. Sci. USA 77:1398-1402).
Moreover some proteins have been identified which have a role in regulation of cell growth. For example many of the cellular protooncogenes are thought to have a normal role in cellular replication which is improperly executed when the protooncogene becomes mutated. The p53 gene is such a protooncogene. Normally the p53 protein appears to inhibit cell growth, however mutant p53 proteins can have an opposite effect upon cell growth, causing uncontrolled cell division and a variety of cancers, especially sarcomas, breast, brain, adrenal cortex, colon, lung and leukemic cancers (Finlay et al. 1989 Cell 57:1083-1093; Ben-David et al. 1991 Cell 66:831-834; and Haber et al. 1991 Cell 64:5-8). Moreover, the p53 protein binds to DNA sites in a sequence-specific manner (Kern et al. 1991a Science 252:1708-1711; Kern et al. 1991b Oncogene 6:131-136). However, the significance of DNA binding by p53 protein relative to the role of p53 protein in cellular replication has not been established.
According to the present invention, cell growth is inhibited by site-specific oligonucleotide binding to DNA. Specifically, the site to which the oligonucleotide binds is a DNA site which can be bound by a protein repressor of cellular replication, e.g. the p53 protein.
Oligonucleotides have recently attracted attention as regulators of nucleic acid biological function. Naturally occurring complementary, or antisense, RNA are used by some cells to control protein expression or plasmid replication. For example, replication of some Escherichia coli plasmids, including the ColE1 plasmid, is regulated by an antisense RNA complementary to an RNA primer of ColE1 DNA replication (Lacatena et al. 1981 Nature 294:623-626; Lin-Chao et al. 1991 Cell 65:1233-1242).
Specific oligonucleotides have also been synthesized and tested as inhibitors of nucleic acid function. For example, splicing of a pre-mRNA transcript essential for Herpes Simplex virus replication has been inhibited with a linear oligonucleotide complementary to an acceptor splice junction (Smith et al., 1986, Proc. Natl. Acad. Sci. USA 83:2787-2791). A linear oligonucleotide has also been used to inhibit protein synthesis of a human immunodeficiency virus (HIV) p24 protein (Agrawal et al. 1988 Proc. Natl. Acad. Sci. USA 85:7079-7083). In another example, linear oligonucleotides were used to inhibit HIV replication in cultured cells. Linear oligonucleotides complementary to sites within or near the terminal repeats of the HIV retroviral genome and within sites complementary to certain splice junctions were most effective in blocking viral replication (Goodchild et al., 1988, Proc. Natl. Acad. Sci. USA 85:5507-5511). Accordingly, the use of oligonucleotides as inhibitors of nucleic acid function has been limited to inhibition of functions such as RNA splicing, protein translation and viral replication via formation of Watson-Crick base pairs between an oligonucleotide and a nucleic acid template. The inhibition of cell growth by oligonucleotide binding has not been demonstrated.
An oligonucleotide binds to a target nucleic acid by forming hydrogen bonds between bases in the target and the oligonucleotide. Common B DNA has conventional adenine-thymine (A-T) and guanine-cytosine (G-C) Watson and Crick base pairs with two and three hydrogen bonds, respectively. The most common bonds that form between two complementary strands of RNA or DNA are Watson-Crick hydrogen bonds. However, other types of hydrogen bonding patterns are known wherein some atoms of a base which are not involved in Watson-Crick base pairing can form hydrogen bonds to another, third, nucleotide. For example, thymine (T) can bind to an A-T Watson-Crick base pair via hydrogen bonds to the adenine, thereby forming a T-AT base triad. Hoogsteen (1959, Acta Crystallography 12:822) first described the alternate hydrogen bonds present in T-AT and C-GC base triads. More recently, G-TA base triads, wherein guanine can hydrogen bond with a central thymine, have been observed (Griffin et al., 1989, Science 245:967-971).
Oligonucleotides have also been observed to bind and inhibit the function of a nucleic acid through non-Watson-Crick hydrogen bonding. For example, Cooney et al. (1988, Science 241:456) disclose a 27-base single-stranded oligonucleotide which bound to a double-stranded nucleic acid via non-Watson-Crick hydrogen bonds. This oligonucleotide inhibited transcription of the human c-myc gene in a cell free, in vitro assay by binding to the c-myc promoter. In a review, Riordan et al. suggest that linear "switchback" oligonucleotides can be used to bind and inhibit the function of both strands of a double stranded nucleic acid target by Watson-Crick binding to one target strand and non-Watson-Crick binding to the other target strand (Riordan et al. 1991 Nature 350:442-443. However, methods for inhibiting cell growth by either non-Watson-Crick or Watson-Crick binding of an oligonucleotide to a chromosomal binding site for a protein repressor of cellular replication are not available in the prior art.
Accordingly, the present invention represents an innovative step forward in the technology of cell cycle control by providing methods for inhibiting cellular replication through Watson-Crick and non-Watson-Crick binding of an oligonucleotide to chromosomal sites normally bound by protein repressors of cellular replication.